This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Agricultural workers, gardeners and homeowners are routinely exposed to the insecticide permethrin. Also, military personnel are exposed to the permethrin when using the DOD Insect Repellent System and over-the-counter lice soaps use permethrin as the active ingredient. A urinary metabolite of permethrin, that is in high abundance and is relatively stable, may be an ideal biomarker of exposure to this pesticide. In addition, the ratio of one metabolite to another may vary, according to the route of administration. The results of this study would be used to identify candidates for the development of a rapid, sensitive immunochemical based analytical method that can be used to routinely monitor human exposure to permethrin. Objectives: The purpose of this study is to determine the human metabolite(s) of permethrin in urine following dermal exposure that are in greatest abundance and are the most stable. Accelerator mass spectrometry is a method for measuring levels of 14C several orders of magnitude more sensitive than liquid scintillation counting. With this high sensitivity we will conduct human metabolism studies at biologically relevant doses. SPECIFIC AIMS: I. Develop an LC/MS method for separation of permethrin and its putative human metabolites. II. Determine the human metabolite profile of permethrin using accelerator mass spectrometry (AMS). III. Develop an immunoassay to the key metabolite identified in Objective II as a biomarker of human exposure to permethrin. METHODOLOGY: For specific aim I, synthesize metabolite standards;develop an HPLC method to separate the putative pyrethroid metabolites using ultraviolet detection;determine the feasibility of an HPLC/mass spectrometry method for analysis of pyrethroid metabolites. For specific aim II, clinical exposure of humans to radiolabeled permethrin dermally and collection of urine, blood and saliva;separation of samples by methods developed in specific aim I and analysis of separated samples by accelerator mass spectrometry;identification of most prevalent metabolite from resultant data. For specific aim III, synthesis of haptens;development of antibodies;use of the haptens and antibodies in the development of an immunoassay for the most prevalent metabolite;validation of immunoassay. EXPECTED PRODUCTS (MILESTONES): Literature review of putative human metabolites of permethrin;small quantities of synthesized metabolites of permethrin;an HPLC method for separating permethrin metabolites in human urine or saliva;identification of the most abundant human metabolite(s) by accelerator mass spectrometry;an immunoassay to detect the targeted human metabolite of permethrin STATUS/RESULTS TO DATE: Literature review has been completed and putative major metabolites identified. All of the major metabolites have been synthesized or acquired. Using the metabolite standards a high performance liquid chromatography method for their analysis has been developed. This method will later be used to identify metabolites found in human urine samples and the liquid chromatography-mass spectrophotometric method used for validation. Using the chemical knowledge from the preparation of metabolites, synthesis of haptens for immunoassay detection of these molecules is complete. Immunoassays for 3-phenoxybenzoic acid, the glycine conjugate of 3-phenoxybenzoic acid, the glycine conjugate of dichlorovinylchrysanthemic acid (DCCA) and the glucuronide conjugate of 3-phenoxybenzyl alcohol (see publication below) have been developed. An assay for free DCCA is in progress (manuscript in preparation). The assay for 3-phenoxybenzoic acid has been adapted to a sensitive, high throughput method (see publication below). Clinical exposures have been completed. All samples have been measured by AMS for total carbon-14. An estimate of the total dose absorbed (for 4 subjects) ranged from 0.06 to 0.27%. The permethrin is eliminated from the blood with a half life of about 12-24 hours hours. The urinary half life averaged 24 hours. Saliva was sampled, but permethrin does not appear to be excreted by that route. Liquid chromatography analysis of the metabolite pattern in urine is underway. Conclusion: The results of this study will be used to identify candidates for the development of a rapid, sensitive immunochemical based analytical method that can be used to routinely monitor human exposure to permethrin. The ability to carefully monitor the presence of absorbed doses of permethrin will be a useful tool to prevent the possibility of human health effects due to permethrin exposure.